Biosynthesis of Thuggacins in Myxobacteria: Comparative Cluster Analysis Reveals Basis for Natural Product Structural Diversity

Abstract

The thuggacins are macrolide antibiotics that are active against Mycobacterium tuberculosis, the causative agent of tuberculosis. Distinct variants of these structures are produced by the myxobacteria Sorangium cellulosum So ce895 and Chondromyces crocatus Cm c5, which differ in side chain structure and modification by hydroxylation. We report here a comparative analysis of the biosynthetic gene clusters in these strains, which reveals the mechanistic basis for this architectural diversity. Although the polyketide-nonribosomal peptide cores of the molecules are highly similar, the underlying biosynthetic machineries exhibit an unexpected degree of divergence. Furthermore, the S. cellulosum gene cluster contains a crotonyl-CoA reductase (CCR) homolog not present in C. crocatus, which likely participates in assembling the unusual hexyl side chain of the So ce895 thuggacins, whereas the distinct hydroxylation pattern may result from variable action of a conserved FMN-dependent monooxygenase. Indeed, inactivation of the monooxygenase gene in C. crocatus resulted in production of both mono- and di-deshydroxy thuggacin derivatives, providing direct evidence for the role of this enzyme in the pathway. Finally, integration of a Tn5-derived npt promotor upstream of the thuggacin cluster in C. crocatus led to a significant increase in thuggacin production.

Mining the Cinnabaramide Biosynthetic Pathway to Generate Novel Proteasome Inhibitors

“…Unexpectedly, it also differs from the hexylmalonyl-CoA-specific TugC-AT3 (Table S1B in the Supporting Information) involved in the biosynthesis of thuggacins-macrolide antibiotics bearing hexyl side chains-from the myxobacterium Sorangium cellulosum So ce895, which are also produced through a NRPS/PKS biosynthetic pathway. [21][22][23] The C-terminal domains of CinA and CinB form the terminal NRPS module of the assembly line, and the highly homologous domain set in the salinosporamide gene cluster is known to activate the nonproteinogenic amino acid b-hydroxy-2'-cyclohexenylalanine. [12,24] Because of the structural similarity of the two compounds, and because the deduced substrate specificity code of the CinB A domain (DLMNVGGV; determined as described previously [25,26] ) showed a significant degree of similarity to that of the SalB A domain (DLLSNGGV), we propose elongation of the cinnabaramide intermediate by extension with b-hydroxy-2'-cyclohexenylalanine (Scheme 2 B).…”

“…[31] Another example has been reported in the biosynthesis of the NRPS/PKS macrolide thuggacin, isolated from Sorangium cellulosum So ce895, in which it was proposed that the hexyl side chain of the compound originates from extension by the unusual PKS extender hexylmalonylCoA, which is thought to be generated from octenyl-CoA by the Ccr homologue TgaD. [21] In the cinnabaramide gene cluster we found a gene (cinF) encoding for a protein showing 90 % sequence identity (at the protein level) to a putative Ccr from Streptomyces hygroscopicus (Accession No. AAR32 675).…”

The Catalytic Diversity of Multimodular Polyketide Synthases: Natural Product Biosynthesis Beyond Textbook Assembly Rules

“…These include biosynthetic enzymes for the myxobacterial metabolites soraphen (24) [99][100][101], myxothiazol (25) [102], melithiazol (26) [103], chondramide (27) [104], thuggacin (28) [105], aurafurone (29) [106], stigmatellin (30) [107], and salinosporamide A (31) [108,109] (Fig. 8).…”

Naturstoffbiotechnologie des Myxobakteriums Chondromyces crocatus

“…2). Durch die Klonierung und Charakterisierung beider Biosynthese-Gencluster ist es gelungen, die molekularen Ursachen für die beobachtete strukturelle Diversität aus den Genen abzulesen [5]. Es ist mittlerweile sogar möglich, basierend auf detaillierten Sequenzanalysen einzelner katalytischer Domänen der Megasynthetasen die vermutliche Stereochemie der Naturstoffe vorherzusagen.…”